CN100509757C - Purification method of *N-L-arginine - Google Patents
Purification method of *N-L-arginine Download PDFInfo
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- CN100509757C CN100509757C CNB2004100895677A CN200410089567A CN100509757C CN 100509757 C CN100509757 C CN 100509757C CN B2004100895677 A CNB2004100895677 A CN B2004100895677A CN 200410089567 A CN200410089567 A CN 200410089567A CN 100509757 C CN100509757 C CN 100509757C
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Abstract
The present invention discloses a method for separating and purifying 15N-L-arginine. The method takes zymotic fluid containing arginine as a raw material, strong acidic resin is used for removing pigThe invention discloses a method for separating and purifying15N-L-arginine, employing the fermentation liquor containing arginine as raw material, removing the coloring matter and foreign matter in tment and impurities in the zymotic fluid, and arginine is separated from other amino acids via ammonia water elution. Then, active carbon is used for decoloring a vacuum concentrated solution containihe fermentation liquor with strong acidic resin, eluting with ammonial solution, separating the arginine from other amino acid, decolourizing the vacuum compression liquor with activated char, vacuum ng the arginine, filter liquor is processed via vacuum concentration and is added in absolute alcohol for purification, and the 15N-L-arginine with purify larger than 98% is prepared. The separating acompressing the filtering liquid, adding compression for refinishing, and getting the 15N-L-arginine with the purity >98%. The separating and purifying method is characterized by simple process, convend purifying method of the present invention has the advantages of simple process and convenient operation, and total extraction yield is higher than 80%. nient operation and more than 80% of the total extraction rate.
Description
Technical field
The present invention relates to a kind of amino acid whose separating and purifying method, particularly a kind of
15N-L-arginic separating and purifying method.
Background technology
L-arginine is the intravital semi-dispensable amino acid of human body and animal, also is a kind of important mesostate of organism ornithine cycle, in fields such as medicine industry, foodstuffs industry important purposes is arranged.
15N-L-arginine is widely used as tracer agent in research fields such as medical science, life sciences owing to the singularity of its structure.
15Biological fermentation process or proteolysis method are generally adopted in N-L-arginic preparation.No matter be which kind of method is produced arginine, all to relate to the problem that arginine and a large amount of impurity are separated.
Prior art is separated arginine and is generally adopted the precipitator method, electroosmose process and ion exchange method.
The precipitator method are to utilize precipitation agent (as phenyl aldehyde, Pentachlorophenol, dodecyl semi-annular jade pendant acid sodium etc.) can make the less mixture of arginine and its generation solubleness and precipitate, thereby reach arginine and the isolating purpose of other amino acid.But the precipitation agent phenyl aldehyde in this method, Pentachlorophenol are toxic compounds, and the discharging of waste liquid behind the extraction arginine can pollute environment; Though and the toxicological harmless of dodecyl semi-annular jade pendant acid sodium, this method complex operation step, dosage is wayward.
15The tracer agent that N-L-arginine is used as industries such as medicine, food should not adopt above three kinds of solvents to carry out purifying.
Though the electroosmose process principle is simple, this method investment is bigger, and the arginine rate of recovery is not high, thereby it is wideless to make this method use.
It is more that ion exchange method is extracted arginic research report, but do not form large-scale production so far yet.At present, there is a large amount of impurity (mainly being pigment and multiple other amino acid) in this method owing to containing in the arginic mixed solution, thereby has influenced exchange capacity, can not separate with other amino acid during wash-out, has influenced total extract yield.
Summary of the invention
Purpose of the present invention is to overcome the deficiency that prior art exists, and provides a kind of new
15N-L-arginic separating and purifying method, this method is directly separated purification from bio-fermented liquid
15N-L-arginine, thus poisonous operation avoided, improved
15N-L-arginic yield.
To achieve these goals, the present invention has adopted following technical scheme: a kind of
15N-L-arginic separating and purifying method is a raw material to contain arginic fermented liquid, takes following steps to separate and purifies:
A, will contain arginic fermented liquid and be adjusted to acidity with oxalic acid, centrifugation goes out supernatant liquor, and supernatant liquor adsorbs with highly acidic resin, uses the ammoniacal liquor desorb, is contained
15N-L-arginic stripping liquid;
B, step a gained is contained
15N-L-arginic stripping liquid vacuum concentration behind dissolved in distilled water, adds activated carbon decolorizing to doing, and suction filtration is contained
15N-L-arginic clear filtrate;
C, step b gained is contained
15N-L-arginic clear filtrate vacuum concentration adds dehydrated alcohol, obtains purified after crystallisation by cooling, filtration, vacuum-drying
15N-L-arginine product.
Described in the step a to be adjusted to tart pH value with oxalic acid be 3-4, described highly acidic resin is that gel strong acid benzene hexene is a resin, described desorb ammonia concn is 0.05～0.2mol/L.
Decolouring described in the step b is carried out under 70～90 ℃.
Temperature during adding dehydrated alcohol described in the step c is controlled at 60～80 ℃, and the temperature during described crystallisation by cooling is controlled at room temperature to-18 ℃, and the temperature during described vacuum-drying is controlled at 50～60 ℃.
Void column flow velocity SV=0.1～1.2% of the resin absorption control centrifuged supernatant described in the step a.
The present invention
15N-L-arginic separating and purifying method makes it compared with prior art owing to adopted above technical scheme, has the following advantages and characteristics:
(1) avoided nontoxic operation, fermented liquid need not to carry out pre-treatment, and adopting gel strong acid benzene hexene is that resin can be removed a large amount of pigments in the fermented liquid, and can make
15N-L-arginine separates with other amino acid (as Threonine, proline(Pro) etc.),
15N-L-arginic single spot yield〉90%, therefore, this technology was both simple, was convenient to operation again.
(2) use dehydrated alcohol as refining solvent, pigment can be stayed in the mother liquor product purity that makes morely 98%, color and luster is snow-white,
15N-L-arginic total extract yield〉80%.
Embodiment
Below by several specific embodiments to the present invention
15N-L-arginic separating and purifying method is further described.
Embodiment 1
What obtain after will fermenting with Corynebacterium glutamicum contains
15N-L-arginic fermented liquid is regulated pH=3.0 with oxalic acid, remove mycelium and throw out in the fermented liquid with the whizzer centrifugation, obtain supernatant liquor, is resin column absorption with supernatant liquor by gel strong acid benzene hexene, control void column flow velocity SV=0.3%, clean resin to neutral with distilled water afterwards, use the ammoniacal liquor desorb of 0.10mol/L then, only contained
15N-L-arginic stripping liquid.Stripping liquid adds an amount of dissolved in distilled water after vacuum concentration is extremely done, add activated carbon decolorizing down at 75 ℃, and the clear filtrate behind the suction filtration is again through vacuum concentration, and the adding dehydrated alcohol is cooled to room temperature then under 70 ℃, in-18 ℃ of freeze overnight,
15N-L-arginine crystallization is separated out, and crystallization is filtered, drained together with mother liquor, with absolute ethanol washing twice, refilters, drains, and obtains
15N-L-arginic crystallisate, then with crystallisate in 60 ℃ of vacuum-dryings, obtain purified
15N-L-arginine product.
Embodiment 2
What obtain after will fermenting with Corynebacterium glutamicum contains
15N-L-arginic fermented liquid is regulated pH=3.6 with oxalic acid, remove mycelium and throw out in the fermented liquid with the whizzer centrifugation, obtain supernatant liquor, is resin column absorption with supernatant liquor by gel strong acid benzene hexene, control void column flow velocity SV=0.8%, clean resin to neutral with distilled water afterwards, use the ammoniacal liquor desorb of 0.15mol/L then, only contained
15N-L-arginic stripping liquid.The stripping liquid vacuum concentration adds an amount of dissolved in distilled water after extremely doing, and adds activated carbon decolorizing down at 80 ℃, and the clear filtrate behind the suction filtration in 80 ℃ of adding dehydrated alcohols, is cooled to room temperature again through vacuum concentration, in-18 ℃ of freeze overnight,
15N-L-arginine crystallization is separated out, and crystallization together with mother liquor-rise to filter, drain, with absolute ethanol washing twice, is refiltered, drains, and obtains
15N-L-arginic crystallisate, then with crystallisate in 50 ℃ of vacuum-dryings, obtain purified
15N-L-arginine product.
Embodiment 3
What obtain after will fermenting with Corynebacterium glutamicum contains
15N-L-arginic fermented liquid is regulated pH=4 with oxalic acid, remove mycelium and throw out in the fermented liquid with the whizzer centrifugation, obtain supernatant liquor, is resin column absorption with supernatant liquor by gel strong acid benzene hexene, control void column flow velocity SV=1.2%, clean resin to neutral with distilled water afterwards, use the ammoniacal liquor desorb of 0.2mol/L then, only contained
15N-L-arginic stripping liquid.The stripping liquid vacuum concentration adds an amount of dissolved in distilled water after extremely doing, and adds activated carbon decolorizing down at 90 ℃, and the clear filtrate behind the suction filtration in 60 ℃ of adding dehydrated alcohols, is cooled to room temperature again through vacuum concentration, in-18 ℃ of freeze overnight,
15N-L-arginine crystallization is separated out, and crystallization is filtered, drained together with mother liquor, with absolute ethanol washing twice, refilters, drains, and obtains
15N-L-arginic crystallisate, then with crystallisate in 55 ℃ of vacuum-dryings, obtain purified
15N-L-arginine product.
In the foregoing description, gained
15The purity of N-L-arginine product reaches more than 98%, always extracts yield and reaches more than 80%.When adopting N atom abundance is 5.31% low abundance feedstock production
15During N-L-arginine, in the products obtained therefrom
15The N abundance reaches more than 5.1%, is 98.90% high abundance feedstock production when employing N atom abundance
15During N-L-arginine, in the products obtained therefrom
15The N abundance reaches more than 98%.
Claims (6)
1. one kind
15N-L-arginic separating and purifying method is characterized in that: to contain arginic fermented liquid is raw material, takes following steps to separate and purifies:
A, will contain arginic fermented liquid and be adjusted to acidity with oxalic acid, centrifugation goes out supernatant liquor, and supernatant liquor adsorbs with highly acidic resin, uses the ammoniacal liquor desorb, is contained
15N-L-arginic stripping liquid;
B, step a gained is contained
15N-L-arginic stripping liquid vacuum concentration behind dissolved in distilled water, adds activated carbon decolorizing to doing, and suction filtration is contained
15N-L-arginic clear filtrate;
C, step b gained is contained
15N-L-arginic clear filtrate vacuum concentration adds dehydrated alcohol, obtains purified after crystallisation by cooling, filtration, vacuum-drying
15N-L-arginine product.
2. as claimed in claim 1
15N-L-arginic separating and purifying method is characterized in that: described in the step a to be adjusted to tart pH value with oxalic acid be 3-4.
3. as claimed in claim 1
15N-L-arginic separating and purifying method is characterized in that: the desorb ammonia concn described in the step a is 0.05～0.2mol/L.
4. as claimed in claim 1
15N-L-arginic separating and purifying method is characterized in that: the decolouring described in the step b is carried out under 70～90 ℃.
5. as claimed in claim 1
15N-L-arginic separating and purifying method, it is characterized in that: the temperature during adding dehydrated alcohol described in the step c is controlled at 60～80 ℃, temperature during described crystallisation by cooling is controlled at room temperature to-18 ℃, and the temperature during described vacuum-drying is controlled at 50～60 ℃.
6. as claimed in claim 1
15N-L-arginic separating and purifying method is characterized in that: void column flow velocity SV=0.1～1.2% of the resin absorption control centrifuged supernatant described in the step a.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100895677A CN100509757C (en) | 2004-12-15 | 2004-12-15 | Purification method of *N-L-arginine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100895677A CN100509757C (en) | 2004-12-15 | 2004-12-15 | Purification method of *N-L-arginine |
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| CN1789236A CN1789236A (en) | 2006-06-21 |
| CN100509757C true CN100509757C (en) | 2009-07-08 |
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| CNB2004100895677A Expired - Fee Related CN100509757C (en) | 2004-12-15 | 2004-12-15 | Purification method of *N-L-arginine |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102001972A (en) * | 2010-10-26 | 2011-04-06 | 广东肇庆星湖生物科技股份有限公司 | Method for separating and extracting L-arginine from fermentation liquor |
| CN102972636A (en) * | 2012-12-03 | 2013-03-20 | 江苏赛奥生化有限公司 | Manufacturing method of feed-grade L-arginine premixing agent |
| CN103667381B (en) * | 2013-12-24 | 2015-09-30 | 山东民强生物科技股份有限公司 | A kind of method improving yield of arginine |
| CN103695489B (en) * | 2013-12-24 | 2015-09-02 | 山东民强生物科技股份有限公司 | A kind of arginine process for refining |
| CN105732436A (en) * | 2016-04-20 | 2016-07-06 | 上海化工研究院 | Method for extracting high-abundance L-arginine-15N4 from high-abundance 15N isotope-labeled L-arginine fermentation liquor |
| CN112479935A (en) * | 2020-12-03 | 2021-03-12 | 江苏优普生物化学科技股份有限公司 | Arginine purification and refining process |
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